The long-term objective of this proposal is to understand intracellular Ca2+ homeostasis in cardiac muscle. Studies with fluorescence digital imaging microscope (FDIM) cellular Ca2+ concentration ([Ca2+]i) in numerous tissues. Moreover, the dynamic nature of intracellular Ca2+ is amplified by the activation of sarcolemmal receptors that are linked to phospholipid breakdown. Although receptor-mediated Ca2+ signal is the subject of intense research interest, little is known about its role in heart. Therefore, we will exploit several techniques to accomplish five specific aims: 1) to complete the experimental work already in progress. 2) To determine quantitatively the spatial distribution of [Ca2+]i in cardiac cells. 3) To improve the time resolution of our FDIM for recording the temporal distribution of [Ca2+]i. 4) To investigate the effects of alpha1- adrenergic receptor, low affinity muscarinic receptor and purinergic receptor activation of [Ca2+]i. 5) To assess the role of protein kinase C (PKC) in modulating L-type Ca2+ channels. Most of the experiments will use single cells from guinea pig and rat ventricles. The [Ca2+]i will be determined with FDIM. The L-type Ca2+ channels will be isolated electrophysiologically by the whole-cell patch-clamp. To correlated [Ca2+]i measurements with the functional aspects of the heart, intracellular Na+ activity and contractility will be measured in papillary muscles. The quantification of [Ca2+]i will be achieved by measuring the ratio values of fura-2 fluorescence at two wavelengths and referring to calibrations obtained from "in vitro" and "in vivo" conditions. The temporal resolution of imaging [Ca2+]i will be improved by a computer- controlled dual beam illumination system. To study the link between the receptors that are coupled to phosphoinostitide turnover and [Ca2+]i, agonists for the alpha1-adrenergic receptor (methoxamine), the low affinity muscarinic receptor (carbachol) and the purinergic receptor (ATP) will be used. To identify the sources of Ca2+ responsible for receptor-mediated [Ca2+]i changes, drugs (e.g. nitrendipine for L-type Ca2+ channels) that inhibit cellular Ca2+ - transport systems will be used. The modulation of L-type Ca2+ channels by PKC will be studied by using phorbol esters, synthetic diacylglycerols, and purified PKC isozymes. These proposed studies will provide information about intracellular Ca2+ homeostasis in cardiac muscle. Because Ca2+ is a key regulator for cardiac function in physiological and pathological states, these studies will broaden our understanding on the fundamental principles of normal and abnormal cardiac excitation and contraction.